HsdS Subunit Research Articles

Structure of HsdS Subunit from Thermoanaerobacter tengcongensis Sheds Lights on Mechanism of Dynamic Opening and Closing of Type I Methyltransferase

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase.

Structural and Functional Analysis of the Engineered Type I DNA Methyltransferase EcoR124INT

SummaryThe Type I R–M system EcoR124I is encoded by three genes. HsdM is responsible for modification (DNA methylation), HsdS for DNA sequence specificity and HsdR for restriction endonuclease activity. The trimeric methyltransferase (M2S) recognises the asymmetric sequence (GAAN6RTCG). An engineered R–M system, denoted EcoR124INT, has two copies of the N-terminal domain of the HsdS subunit of EcoR124I, instead of a single S subunit with two domains, and recognises the symmetrical sequence GAAN7TTC. We investigate the methyltransferase activity of EcoR124INT, characterise the enzyme and its subunits by analytical ultracentrifugation and obtain low-resolution structural models from small-angle neutron scattering experiments using contrast variation and selective deuteration of subunits.

Structural model for the multisubunit Type IC restriction-modification DNA methyltransferase M.EcoR124I in complex with DNA

Recent publication of crystal structures for the putative DNA-binding subunits (HsdS) of the functionally uncharacterized Type I restriction–modification (R-M) enzymes MjaXIP and MgeORF438 have provided a convenient structural template for analysis of the more extensively characterized members of this interesting family of multisubunit molecular motors. Here, we present a structural model of the Type IC M.EcoR124I DNA methyltransferase (MTase), comprising the HsdS subunit, two HsdM subunits, the cofactor AdoMet and the substrate DNA molecule. The structure was obtained by docking models of individual subunits generated by fold-recognition and comparative modelling, followed by optimization of inter-subunit contacts by energy minimization. The model of M.EcoR124I has allowed identification of a number of functionally important residues that appear to be involved in DNA-binding. In addition, we have mapped onto the model the location of several new mutations of the hsdS gene of M.EcoR124I that were produced by misincorporation mutagenesis within the central conserved region of hsdS, we have mapped all previously identified DNA-binding mutants of TRD2 and produced a detailed analysis of the location of surface-modifiable lysines. The model structure, together with location of the mutant residues, provides a better background on which to study protein–protein and protein–DNA interactions in Type I R-M systems.

Tetra-amino-acid tandem repeats are involved in HsdS complementation in type IC restriction-modification systems.

All known type I restriction and modification (R-M) systems of Escherichia coli and Salmonella enterica belong to one of four discrete families: type IA, IB, IC or ID. The classification of type I systems from a wide range of other genera is mainly based on complementation and molecular evidence derived from the comparison of the amino acid similarity of the corresponding subunits. This affiliation was seldom based on the strictest requirement for membership of a family, which depends on relatedness as demonstrated by complementation tests. This paper presents data indicating that the type I NgoAV R-M system from Neisseria gonorrhoeae, despite the very high identity of HsdM and HsdR subunits with members of the type IC family, does not show complementation with E. coli type IC R-M systems. Sequence analysis of the HsdS subunit of several different potential type IC R-M systems shows that the presence of different tetra-amino-acid sequence repeats, e.g. TAEL, LEAT, SEAL, TSEL, is characteristic for type IC R-M systems encoded by distantly related bacteria. The other regions of the HsdS subunits potentially responsible for subunit interaction are also different between a group of distantly related bacteria, but show high similarity within these bacteria. Complementation between the NgoAV R-M system and members of the EcoR124 R-M family can be restored by changing the tetra-amino-acid repeat within the HsdS subunit. The authors propose that the type IC family of R-M systems could consist of several complementation subgroups whose specificity would depend on differences in the conserved regions of the HsdS polypeptide.

Domain structure and subunit interactions in the type I DNA methyltransferase M. EcoR124I

The type IC DNA methyltransferase M. EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM) 2 is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM) 2 complex is able to bind to both the EcoR124I DNA recognition sequence GAAN 6RTCG and a symmetrical DNA sequence GAAN 7TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM) 2 -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3′ and 5′ sides of the recognition sequence bound by the (S3/HsdM) 2 complex.

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M 2S 1. Here, we analysed the impact on the restriction and modification activities of the change Trp 212 → Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.

Characterization of a Novel Plasmid-Encoded HsdS Subunit, S.LlaW12I, from Lactococcus lactis W12

A novel type I restriction-modification specificity subunit, S.LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp. cremoris W12. Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits. Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC. Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit.

Localization of the Type I Restriction–Modification Enzyme EcoKI in the Bacterial Cell

To localise the type I restriction–modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties.

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42 degrees C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA-protein and protein-protein interactions, respectively.

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser 340Phe) and Sts2 (Ala 204Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30°C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42°C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA–protein and protein–protein interactions, respectively.

The DNA recognition subunit of the type IB restriction-modification enzyme EcoAI tolerates circular permutions of its polypeptide chain

The DNA specificity subunit (HsdS) of type I restriction-modification enzymes is composed of two independent target recognition domains and several regions whose amino acid sequence is conserved within an enzyme family. The conserved regions participate in intersubunit interactions with two modification subunits (HsdM) and two restriction subunits (HsdR) to form the complete endonuclease. It has been proposed that the domains of the HsdS subunit have a circular organisation providing the required symmetry for their interaction with the other subunits and with the bipartite DNA target. To test this model, we circularly permuted the HsdS subunit of the type IB R-M enzyme EcoAI at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circularly permuted variants of HsdS that had termini located at equivalent positions in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity, suggesting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stable trimeric complex with HsdM, M 2S 1, which was fully active as a DNA methyltransferase. Gel electrophoretic mobility shift assays revealed that the HsdS protein alone was not able to form a specific complex with a 30-mer oligoduplex containing a single EcoAI recognition site. However, addition of stoichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular organisation of domains of the HsdS subunit. In addition, they suggest a possible role of HsdM subunits in the formation of this structure.

The effect of recA mutation on the expression of EcoKI and EcoR124I hsd genes cloned in a multicopy plasmid.

Type I restriction-modification (R-M) endonucleases are composed of three subunits--HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of cloned EcoKI and EcoR124I hsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wt hsd genes in a multicopy plasmid in Escherichia coli recA+ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the cloned hsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation in E. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.

Combinational variation of restriction modification specificities in Lactococcus lactis.

Three genes coding for a type I R-M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403. In addition, plasmids were found that encode only the HsdS subunit that directs R-M specificity. The presence of these plasmids in IL1403 conferred a new R-M phenotype on the host, indicating that the plasmid-encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits. Such combinational variation of type I R-M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.

Expression and characterisation of the N-terminal fragment of the HsdS subunit of M.EcoR124I.

The type IC modification methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two copies of the modification subunit, HsdM, and a single DNA specificity subunit, HsdS. Studies to date have been largely restricted to the HsdM subunit or the intact methyltransferase, since the HsdS subunit is insoluble when expressed independently of HsdM. Using PCR, we have cloned and expressed 13 fragments of the gene for the HsdS subunit, including the sequences encoding each of the variable and conserved domains and various combinations of these. Only two of these fragments were found to be soluble, a 8.6 kDa fragment (S11) comprising the central conserved domain and a 25 kDa N-terminal fragment (S3) containing the N-terminal variable domain and the central conserved domain. Analysis of the larger of these fragments by gel retardation shows that the protein binds DNA in the presence of HsdM at a subunit stoichiometry of 1:1. Gel filtration and CD spectroscopy indicate that the protein is monomeric and predominantly alpha-helical.

DNA binding and subunit interactions in the type I methyltransferase M.EcoR124I.

The type I DNA methyltransferase M.EcoR124I consists of two methylation subunits (HsdM) and one DNA recognition subunit (HsdS). When expressed independently, HsdS is insoluble, but this subunit can be obtained in soluble form as a GST fusion protein. We show that the HsdS subunit, even as a fusion protein, is unable to form a discrete complex with its DNA recognition sequence. When HsdM is added to the HsdS fusion protein, discrete complexes are formed but these are unable to methylate DNA. The two complexes formed correspond to species with one or two copies of the HsdM subunit, indicating that blocking the N-terminus of HsdS affects one of the HsdM binding sites. However, removal of the GST moiety from such complexes results in tight and specific DNA binding and restores full methylation activity. The results clearly demonstrate the importance of the HsdM subunit for DNA binding, in addition to its catalytic role in the methyltransferase reaction.

Generation of new DNA binding specificity by truncation of the type IC EcoDXXI hsdS gene.

The hsdS subunit of a type IC restriction-modification enzyme is responsible for the enzyme's DNA binding specificity. Type I recognition sites are characterized by two defined half-sites separated by a non-specific spacer of defined length. The hsdS subunit contains two independent DNA binding domains, each targeted towards one DNA half-site. We have shown previously that the 5' half of hsdS can code for a functional substitute of the full-length hsdS. Here we demonstrate that the 3' half of the gene, when fused to the appropriate transcriptional and translational start signals, also codes for a peptide which imparts DNA binding specificity to the enzyme. About half the natural hsdS size, the mutant peptide contains a single DNA recognition domain flanked by one copy of each internal repeat found in the full-length hsdS. Deletion of either repeat sequence results in loss of activity. Like the 5' hsdS mutant, the 3' mutant recognizes an interrupted palindrome, GAAYN(5)RTTC, suggesting that two truncated subunits participate in DNA recognition. Co-expression of the 5' hsdS mutant and the 3' hsdS mutant along with hsdM regenerates the wild-type methylation specificity. Thus, there is a free assortment of subunits in the cell.

Surface Labelling of the Type I Methyltransferase M. EcoR124I Reveals Lysine Residues Critical for DNA Binding

The type IC methyltransferase M. EcoR124I consists of a specificity subunit (HsdS) and two methylation subunits (HsdM). Using chemical modification, we have investigated the accessibility of lysine residues in the free enzyme and in the complex with its DNA recognition sequence. A total of 41 of the 109 lysine residues in the enzyme are susceptible to modification, of which 19 are located in the HsdS subunit and 11 in each of the two HsdM subunits. DNA binding results in extensive protection of lysine residues in the HsdS subunit, while those in the HsdM subunit are only protected weakly. The DNA binding activity of the methylase is abolished when a small fraction of the accessible lysine residues are modified. Peptide mapping and N-terminal sequencing has been used to locate the rapidly modified lysine residues in HsdS that are critical for DNA binding. Highly modified residues (K297, K261 and K327) are found in the C-terminal variable domain that is responsible for DNA recognition, but others (K196, K203 and K210) are found in the conserved regions that had not previously been implicated in DNA binding.

Probing the Domain Structure of the Type IC DNA Methyltransferase M. EcoR124I by Limited Proteolysis

Limited proteolysis has been used to probe the domain structure of the type I DNA methyltransferase M. EcoR124I. Trypsin digestion of the methyltransferase generates two fragments derived from the HsdS subunit, a 28 kDa N-terminal domain and a 19 kDa C-terminal domain, leaving the HsdM subunit intact. Extensive digestion by chymotrypsin, however, removes 59 amino acid residues from the N terminus of the HsdM subunit to leave a 52 kDa C-terminal domain. Binding of the cofactor S-adenosyl methionine has no appreciable effect on the rate of cleavage, but binding of a 30 bp DNA duplex containing the cognate recognition sequence confers almost total protection. Following trypsin cleavage of the methyltransferase, a stable proteolytic product is produced which has been purified for biochemical characterisation. The trypsinised enzyme is shown to be a multimeric complex containing two intact HsdM subunits and both fragments of the HsdS subunit, consistent with the circular model proposed for the organisation of domains in the specificity subunit in type IC methyltransferases. Gel retardation studies show that the proteolysed enzyme still retains DNA binding activity, but its specificity for the DNA recognition sequence is dramatically reduced. f2 f2 Abbreviations used: R-M, restriction-modification; DFP, di-isofluropropanol; HPLC, high pressure liquid chromatography.

The HsdS polypeptide of the type IC restriction enzyme EcoR124 is a sequence-specific DNA-binding protein.

The HsdS and HsdM polypeptides of the type IC restriction enzyme EcoR124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence-specific recognition of DNA by this enzyme. The HsdS polypeptide alone is able to bind to DNA in a sequence-specific manner. In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA.

Mutation in the specificity polypeptide of the type I restriction endonuclease R · EcoK that affects subunit assembly

We describe the isolation and characterization of a temperature-sensitive mutation within the hsdS gene of the type I restriction and modification system EcoK. This mutation appears to affect the ability of the HsdR subunit to interact with the HsdS subunit when forming an active endonuclease. We discuss the possibility that this mutant, together with another mutation described previously, may define a discontinuous domain, involved in protein-protein interactions, within the HsdS polypeptide.